The long term goal of this project is to characterize neurotransmitter receptor-mediated information transduction, and its regulation, across neuronal membranes. The primary receptor systems under investigation are those for dopamine. In order to characterize these receptors at the biochemical and molecular levels, and study their regulation, there are two interrelated lines of research being performed: 1) investigation of the cell biology, function and regulation of the receptors at the protein level; and 2) the molecular cloning of the receptor cDNAs/genes and investigation of receptor structure, pharmacology and regulation in cultured cell lines and transgenic mice. The role of protein phosphorylation in regulating D-1 receptor function was examined using mutagenesis and metabolic labeling techniques. We had previously found that when four potential protein kinase A (PKA) phosphorylation sites in the receptor were eliminated, the mutant receptor exhibited slower kinetics of desensitization subsequent to dopamine exposure. Further analyses of single mutated receptors, in which only one of the four sites is modified, reveals that Thr-268 in the 3rd cytoplasmic loop of the receptor is primarily responsible for the effects of PKA. We have also mutated potential G protein-coupled receptor kinase (GRK) phosphorylation sites within the 3rd cytoplasmic loop of the receptor. Several of these mutant receptors demonstrate delayed/reduced agonist-induced desensitization. Using C6 cells stably transfected with an epitope tagged D-1 receptor, we have been able to directly demonstrate agonist-induced phosphorylation of the receptor protein. The phosphorylation status of mutated D-1 receptors is presently under investigation. The epitope-tagged D-1 receptor has also been visualized in transfected cells using both fluorescence and confocal microscopy and we are investigating "internalization" motifs in the receptor protein using mutagenesis approaches. Similar regulatory work has also begun on the D-2S and D-2L receptor isoforms. Work also continued on cloning a third "D-1 like" receptor which stimulates phosphatidylinositol (PI) turnover and calcium mobilization. Homozygous D-5 receptor knock-out mice were generated and determined to be viable, fertile and neurologically intact. Preliminary behavioral experimentation suggests that the animals exhibit greater exploratory activity in an open field test which is not due to reduced anxiety. The animals also exhibit superior motor learning skills as assessed with the rotarod test. These animals are currently undergoing additional physiological/behavioral characterization.